Breast cancer is the most common disease and leading cause of lethality in women. The identification and cloning of the BRCA1 and BRCA2 genes have demonstrated that mutations in these two genes account for almost all of the families with multiple cases of breast and ovarian cancer. To understand the biological function of these genes, mutations that result in loss of function have been generated in mice. These studies have demonstrated the involvement of these two genes in cell proliferation and normal development and also in the Rad51-mediated DNA recombination repair machinery. Mutation in the Brca1 or Brca2 genes may result in defect in DNA repair/monitor system causing accumulation of mutation in growth control genes, leading to tumorigenesis. One of the goals of our research is to conduct a functional dissection of the Brca1 and Brca2 protein and to determine other components of the pathway(s) in which these function. To achieve this, we are generating subtle mutations in regions of the gene encoding these two proteins to study their phenotypic effect. To generate these multiple alleles, we are using bacterial artificial chromosomes (BACs). We have identified BAC clones that contain the functional Brca1 and Brca2 gene, as they can rescue the embryonic lethality associated with mutation in these genes. We are using BACs with mutation in these genes to generate transgenic mice on a Brca1 or Brca2 mutant background. We are manipulating the BACs by using the bacteriophage recombination system. We have developed a rapid method to manipulate BACs to generate deletions, insertions and single base changes using oligonucleotides as targeting vectors obviating the use of selection markers. We have developed a PCR-based approach to identify the correctly targeted clone. In human cancer patients, in addition to mutations that clearly disrupt the gene, several missense mutations have been identified in the BRCA1 and BRCA2 genes. The functional significance of these mutations is unknown. These may result in an unstable protein, alter a subset of its function, or represent a rare polymorphic variant. Our aim is to generate missense mutations in murine Brca1 and Brca2 genes and study their functional significance using the BAC transgenic approach. We have examined the possibility of using human gene in mice to generate missense mutations. We have generated humanized mice using human BRCA1 as well as the BRCA2 gene. These transgenic mice are able to rescue to embryonic lethality associated with mutation in the endogenous Brca1 or Brca2 genes. We are generating missense mutations in the human gene to examine their functional significance. Such mutations will serve as valuable tool for functional dissection of these genes.